Seasonal nitrogen and carbon concentrations in white,brown and woody fine roots of sugar maple (Acer saccharum Marsh)

Small diameter (<1.0-mm) Acer saccharum Marsh roots were separated into white, brown and woody development state classes and analyzed for total N and C concentrations in April, July and October of 1988. White roots had greater concentrations of N and C than either brown or woody roots at each sampling date, and the N concentration of brown roots was consistently greater than that of woody roots. There were no temporal changes in N concentrations in any of the roots. C was slightly elevated in mid-summer in all three classes of roots. The data suggest the possible existence of an N translocation mechanism in ageing and developing fine roots. More research should be undertaken to establish the mechanisms of N loss in developing fine roots.     

Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.     

Applications of minirhizotrons to understand root function in forests and other natural ecosystems

Minirhizotrons have proved useful to understand the dynamics and function of fine roots. However, they have been used comparatively infrequently in forests and other natural plant communities. Several factors have contributed to this situation, including anomalous root distributions along the minirhizotron surface and the difficulty of extracting data from minirhizotron images. Technical and methodological advances have ameliorated some of these difficulties, and minirhizotrons have considerable potential to address some questions of long standing interest. These questions include more fully understanding the role of roots in carbon and nutrient cycling, rates of root decomposition, responses to resource availability and the functional significance of interactions between plant roots and soil organisms. Maximizing the potential for minirhizotrons to help us better understand the functional importance of fine roots in natural plant communities depends upon using them to answer only those questions appropriate to both their inherent strengths and limitations.     

Urinary 6-Sulfatoxymelatonin Cycle-To-Cycle Variability

For either clinical or research purposes, the timing of the nocturnal onset in production of the urinary melatonin metabolite 6-sulfatoxymelatonin (UaMT6s-onset), has been proposed as a reliable and robust marker of circa-dian phase. However, given that most circadian rhythms show cycle-to-cycle variability, the statistical reliability of phase estimates obtained from a single study using UaMT6s-onset remains to be determined. Following 2 weeks of sleep diary and wrist actigraphy, 15 young, healthy good sleepers participated in four UaMT6s sampling sessions spaced 1 day apart. During the sampling sessions subjects remained indoors under low light conditions and hourly urine samples were collected from 19:00 to 02:00 h. Samples were subsequently assayed for UaMT6s using standard radioimmunographic techniques. UaMT6s-onset was determined by the time at which melatonin production exceeded the average of three proceeding trials by 100%. Sleep onset times were derived from sleep diary and actigraphic measures taken before the melatonin collection nights. We found that there was no significant variation between nights in group mean UaMT6s-onset times, and intraindividual variability was small. In addition, UaMT6s-onset times were highly and significantly correlated between nights (grand mean r = 0.804). Our results suggest that within 95% confidence interval limits, individual UaMT6s-onset estimates obtained from a single night UaMT6s-onset study can be used to predict subsequent UaMT6s-onset times within ±97 min. A close temporal relationship was also found between the timing of UaMT6s-onset and sleep onset. Overall, our results suggest that under entrained conditions single-session UaMT6s-onset studies can provide reliable individual UaMT6s-onset phase estimates and that the protocol described in this study is a practical and noninvasive methodology. (Chronobiology International, 13(6), 411-421, 1996)     

Secretion of a heat-stable fungalβ-glucanase from transgenic,suspension-cultured barley cells

Transgenic plant cell cultures have a potential for production and secretion of important proteins and peptides. To assess the possibilities of using a stable barley suspension culture for secretion of heterologous proteins in active form, we expressed the cDNA of the thermostable-glucanase (EGI) ofTrichoderma reesei in barley suspension cells. The cDNA coding for EGI and its signal sequence was placed under the control of the CaMV 35S promoter and the construction was transferred to the cells by particle bombardment. Stably transformed lines were obtained by selecting for a cotransformed antibiotic resistance marker. The expression of EGI cDNA led to accumulation of EGI in the culture medium, as shown by analysis with EGI-specific antibodies. Enzymatic assays confirmed that the EGI secreted by the suspension cells retained its activity and thermostable character. Furthermore, it was shown that the enzyme produced by the transgenic suspension culture could be used for degradation of soluble-glucans during mashing.     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.     

A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli.

A 1.5-kb genomic fragment isolated from Streptomyces avermitilis that directs the synthesis of a brown pigment in Escherichia coli was characterized. Since pigment production in recombinant E. coli was enhanced by the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated with melanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitilis was unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototype of melanin genes in Streptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single open reading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic and eukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressed in E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), which is the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brown pigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients. Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations of this acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, the production of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitilis gene encoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated in the medium by this clone.     

H-2T24 and Pema T24: orthologous expressed MHC class Ib genes from mouse and Peromyscus maniculatus

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U03104 and L22338.     

Fertile transgenic barley by particle bombardment of immature embryos

Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T₀), and four of the 90 T₀ spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T₁). The four transgenic shoots of Toivo (the T₀ plant) produced 25 plantlets as T₁ progeny. Altogether fifteen of these T₁ plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T₂ progeny.